Several human proteins are known to form amyloid fibrils, and these fibrils are associated with several devastating diseases, including Alzheimer's, Parkinson's, type II diabetes, and dialysis-related amyloidosis (DRA). One of these proteins, human beta-2- microglobulin (beta2m), can form amyloid fibrils in the presence of Cu(II). The fibrils formed by beta2m are the main pathogenic process underlying DRA. Like other amyloid systems, beta2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While many general aspects of amyloid formation are understood, molecular-level information for the early stages of almost all amyloid forming reactions is only starting to emerge. This information, though, is critical for the rational development of therapeutics against amyloid diseases like DRA. We intend to obtain amino acid-level information about the unfolding and oligomerization of beta2m that precedes its fibril formation, with a particular emphasis on the unique role that Cu(II) plays in inducing this reaction. We will also investigate the molecular basis of several inhibitors that show promise for disrupting beta2m amyloid formation. To obtain the desired insight, we have three main aims. First, we will explore a combination of covalent labeling and hydrogen/deuterium exchange with mass spectrometric detection to obtain a detailed picture of the structural changes that betam and its oligomers undergo prior to amyloid formation. Second, we will develop and apply new labeling methods in conjunction with mass spectrometry to study metal-protein interactions with the goal of understanding the unique role that Cu(II) plays in beta2m amyloid formation. Third, our new labeling approaches will be applied to understand the molecular basis of three inhibitors of beta2m amyloid formation. Our prior work has revealed that Cu(II) destabilizes beta2m, allowing it to form oligomers prior to amyloid fibrils. We hypothesize that Cu(II) initiate the amyloid reaction by repositioning several regions of the protein and that Cu(II)'s coordination chemistry enables these structural and oligomeric changes in a way that is unique among transition metals. Our mass spectrometry-based methods will allow us to test this hypothesis, and along with the proposed inhibitor studies, we will obtain a deeper understanding of beta2m amyloid formation, which will facilitate the design of better therapeutics against DRA. Moreover, we expect that the techniques developed here will be applicable to other amyloid systems.

Public Health Relevance

The formation of protein amyloid fibrils is associated with about 20 human diseases, including Alzheimer's, Parkinson's, and Dialysis-Related Amyloidosis (DRA), but the molecular details of how these amyloids begin to form are mostly unknown. We intend to develop and apply new mass spectrometry based measurement tools that will provide the molecular details necessary to understand the amyloid formation of beta-2-microglobulin (beta2m), which is the protein implicated in DRA. The information that we uncover about beta2m should better inform efforts to design therapeutics against DRA but will also give insight into protein amyloid formation in general.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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University of Massachusetts Amherst
Schools of Arts and Sciences
United States
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Zhang, Zhe; Vachet, Richard W (2015) Kinetics of Protein Complex Dissociation Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry. Anal Chem 87:11777-83
Zhang, Zhe; Browne, Shaynah J; Vachet, Richard W (2014) Exploring salt bridge structures of gas-phase protein ions using multiple stages of electron transfer and collision induced dissociation. J Am Soc Mass Spectrom 25:604-13
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Dong, Jia; Callahan, Katie L; Borotto, Nicholas B et al. (2014) Identifying Zn-bound histidine residues in metalloproteins using hydrogen-deuterium exchange mass spectrometry. Anal Chem 86:766-73
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Zhou, Yuping; Vachet, Richard W (2013) Covalent labeling with isotopically encoded reagents for faster structural analysis of proteins by mass spectrometry. Anal Chem 85:9664-70
Zhou, Yuping; Vachet, Richard W (2012) Diethylpyrocarbonate labeling for the structural analysis of proteins: label scrambling in solution and how to avoid it. J Am Soc Mass Spectrom 23:899-907
Zhou, Yuping; Vachet, Richard W (2012) Increased protein structural resolution from diethylpyrocarbonate-based covalent labeling and mass spectrometric detection. J Am Soc Mass Spectrom 23:708-17
Dong, Jia; Vachet, Richard W (2012) Coordination sphere tuning of the electron transfer dissociation behavior of Cu(II)-peptide complexes. J Am Soc Mass Spectrom 23:321-9
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